A General Method for Fusion of the Escherichia coli lacZ Gene to Chromosomal Genes in Bacillus subtilis
- 1 November 1986
- journal article
- research article
- Published by Microbiology Society in Microbiology
- Vol. 132 (11), 2953-2966
- https://doi.org/10.1099/00221287-132-11-2953
Abstract
A series of plasmids has been constructed that can be used to fuse the .beta.-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or ''cartridge'' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as .vphi.105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.This publication has 5 references indexed in Scilit:
- Use of Integrational Plasmid Vectors to Demonstrate the Polycistronic Nature of a Transcriptional Unit (spoIIA) Required for Sporulation of Bacillus subtilisMicrobiology, 1984
- Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination.Proceedings of the National Academy of Sciences, 1983
- Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloningGene, 1982
- Nucleotide sequences that signal the initiation of transcription and translation inBacillus subtilisMolecular Genetics and Genomics, 1982
- Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosomeJournal of Bacteriology, 1980